Effect of antioxidants (sodium pyruvate and catalase) on post thaw motility

Freezing of the semen in liquid nitrogen is the only method for long term storage of fertile spermatozoa. The procedure involves different steps which are harmful to spermatozoa and consequently reduce their quality and fertility. In many animal species the addition of antioxidants to semen before freezing was found to have positive effect on the quality and fertility of frozen/thawed (F/T) spermatozoa. As in rams there are contradictory results in the literature concerning the influence of specific antioxidants on sperm quality of F/T spermatozoa, the present study was performed to evaluate the effects of two different antioxidants on post thaw motility, viability and DNA integrity of F/T ram spermatozoa. Ejaculates from six crossbreed rams were frozen according to the standard procedure after two step dilution with modified Tris-egg yolk extender. The second extender, which contained 14% of glycerol, was added to the semen at 5°C. Ejaculates were divided into three parts and extended with diluents that were either supplemented with 5mM sodium pyruvate (group 1), 20IU/ml catalase (group 2), both antioxidants together (group 3) or contained no antioxidants (control group). Diluted samples were loaded into 0.5ml straws and frozen in nitrogen vapour, 4cm above the liquid nitrogen. Frozen samples were kept in liquid nitrogen for at least two month before thawing and analysis. Frozen straws were thawed in water bath at 37°C for 17s. Motility and viability (Viadent®) of the F/T samples were analysed with Hamilton Thorne Biosciences, Version 12.3, after incubation in water bath at 37°C for 10min, 6,12 and 24h. The percentages of sperm showing a high DNA fragmentation (DFI%) of F/T spermatozoa was analysed 5min and 3h after thawing by using the flow cytometric sperm chromatin structure assay (SCSA™). Statistical analyses were performed with Sigma Stat (Version 3.10). Statistical difference between the groups was tested with Kruskal-Wallis One Way Analysis of Variance on Ranks. Values were considered to be statistically significant when p0.44). Analysis of SCSA™ also revealed no difference (1.7 ± 1.2% vs. 1.6 ± 0.9% vs. 1.7 ± 1.0 vs. 1.6 ± 1.1% and 1.8 ± 1.3% vs. 1.7 ± 1.0% vs. 1.9 ± 1.2 vs. 1.7 ± 1.3% for group 1 vs. 2 vs. 3 vs. control respectively for 0 and 3h after F/T) (p>0.06) the p-value is rather low and indicates that there is somewhere a difference by trend (0.05<p<0.10) in DFI%-values between groups with antioxidants compared to control group 0 and 3h after thawing. These results indicate no positive effect of pyruvate and catalase on motility, viability and chromosome integrity of F/T ram spermatozoa

The effect of Equex STM in freezing media on post thaw motility, viability and dna integrity of frozen - thawed ram spermatozoa

In this study we investigated the effect of Equex STM® on quality and in-vitro survival of ram spermatozoa frozen in Tris egg yolk based extender. Ejaculates from 6 crossbreed rams were frozen according to the standard procedure after two step dilution with Tris-egg yolk extender (1). The second extender, added to the semen at 5°C, contained 14% of glycerol and was supplemented with detergent 0.75% Equex STM® (group OEP) or contained no detergent (control group). After thawing the samples were incubated in a water bath at 37°C and analysis were performed 10 minutes, 6, 12 and 24 hours later. Motility and the viability (Viadent®) of the semen were analysed with Hamilton Thorne Biosciences, Version 12.3 and membrane integrity with HOS (hypoosmotic swelling test). DNA fragmentation (DFI %) of F/T spermatozoa was analyzed 10 minutes and 3 hours after thawing using sperm chromatin structure assay (SCSATM). The sperm membrane integrity was analysed 15 minutes and 3 hours after thawing by Sybr-14/PI test. Percentage of motile spermatozoa was significantly higher in OEP group in comparison to control group at 0, 6, 12 and 24 h (P<0.001). Viability of spermatozoa was significantly higher (P<0.001) in OEP compared to control group in all analysed times after thawing. Percentage of HOS positive spermatozoa was significantly higher in OEP compared to control group respectively for 0 (P=0.001), 6 (P=<0.001), 12 and 24 h (P=0.002) after thawing